Silk microspheres for encapsulation and controlled release

ABSTRACT

A method was developed to prepare silk fibroin microspheres using lipid vesicles as templates to efficiently load therapeutic agents in active form for controlled release. The lipids are subsequently removed through the use of a dehydration agent, such as methanol or sodium chloride, resulting in β-sheet structure dominant silk microsphere structures having about 2 μm in diameter. The therapeutic agent can be entrapped in the silk microspheres and used in pharmaceutical formulations for controlled-release treatments.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/778,402 filed on Feb. 27, 2013, which is acontinuation application of U.S. patent application Ser. No. 12/442,595filed on Jul. 7, 2009 and now abandoned, which is a 35 U.S.C. §371National Stage Application of International Application No.PCT/US2007/020789 filed on Sep. 26, 2007, which designates the U.S. andwhich claims benefit under 35 U.S.C. §119(e) of U.S. ProvisionalApplication No. 60/847,100 filed on Sep. 26, 2006, the contents of eachof which are incorporated herein by reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. DE016525awarded by the National Institutes of Health. The Government has certainrights in the invention.

FIELD OF INVENTION

This invention relates to silk fibroin microspheres prepared by mixingsilk fibroin with lipids to form microspheres capable of encapsulatingtherapeutic drugs and releasing the drugs in a controlled manner.

BACKGROUND OF INVENTION

Controlled drug release involves a combination of a polymer matrix withbioactive drugs such that the drugs can be delivered in a predictablemanner. Polymeric materials, including biodegradable synthetic polymerssuch as poly(D,L-lactide-co-glycolide) (PLGA) and natural polymer suchas collagen and alginate have been used as drug delivery matrices. Thesepolymer matrices function in many ways as an artificial extracellularmatrix (ECM) to stabilize encapsulated proteins, such as growth factors.See Jiang et al., “Biodegradable poly(lactic-o-glycolic acid)microparticles for injectable delivery of vaccine antigens,” Adv. DrugDeliv. Rev. 57 (2005) 391-410; see also Wee et al., “Protein releasefrom alginate matrices,” Adv. Drug Deliv. Rev. 31 (1998) 267-285.

The release of encapsulated protein drugs are controlled by both passivediffusion of protein drugs and degradation of polymer matrices.Encapsulation and controlled release are of particular importance forprotein drugs with short half-lives when free in solution, and forreduced systemic toxicity. However, preservation of biological activityof incorporated protein drugs in a polymer matrix and control ofsubsequent release remain major challenges.

Silk fibroin has a long history in clinical applications used as suturethreads, and now it is finding new and important applications in thetissue-engineering field as a scaffold support for the growth ofartificial tissues such as bone and cartilage. Recently, the use of silkfibroin for controlled drug delivery has been explored with electrospunsilk fiber mats that encapsulated bone morphogenetic protein 2 (BMP-2).See Li et al., “Electrospun silk-BMP-2 scaffolds for bone tissueengineering,” Biomaterials 27 (2006):3115-3124. Hoffman investigated theencapsulation and release of different proteins such as horseradishperoxidase (HRP) and lysozyme from silk films and the correlationbetween silk crystallinity that were induced by methanol and proteinrelease behaviors. It was found that high silk crystallinity couldsignificantly retard the release of encapsulated proteins. See Hofmannet al., “Silk fibroin as an organic polymer for controlled drugdelivery,” J. Control Release 111 (2006):219-227.

Thus, silk fibroin holds great promise for controlled drug delivery dueto its unique structure and crystallinity properties as well as theother advantages discussed above. Silk microspheres can be fabricatedusing physical methods such as spray-drying, however, harsh conditionssuch as high temperature have prohibited their uses as a protein drugdelivery carrier. See Hino et al., “Change in secondary structure ofsilk fibroin during preparation of its microspheres by spray-drying andexposure to humid atmosphere,” J. Colloid Interface Sci. 266 (2003)68-73. In addition, conventional microspheres typically have a largesize (above 100 μm), making them less useful as encapsulation vehiclesfor many of the smaller drug molecules.

Accordingly, what is needed in the art is a way to prepare silk fibroinmicrospheres under mild conditions so that protein drugs and othertherapeutic agents can be encapsulated in the microspheres and releasedin their active forms. This invention answers that need.

SUMMARY OF INVENTION

One embodiment of this invention relates to a method of preparing silkfibroin microspheres. The method involves (a) mixing a silk fibroinsolution with a lipid composition; (b) lyophilizing the mixture; (c)combining the lyophilized material with a dehydration medium for asufficient period of time to at least partially dehydrate the silkfibroin solution and induce β-sheet structures in the silk fibroin; and(d) removing at least a portion of the lipids to form silk fibroinmicrospheres.

Another embodiment of this invention relates to a drug deliverycomposition comprising a therapeutic agent encapsulated in crosslinkedsilk fibroin microspheres, wherein the microspheres contain lipidcomponents.

Another embodiment of this invention relates to a method ofencapsulating a biomaterial in silk fibroin microcapsules. The methodcomprises (a) mixing a solution comprising silk fibroin and abiomaterial with a lipid composition; (b) lyophilizing the mixture; (c)combining the lyophilized material with a dehydration medium for asufficient period of time to at least partially dehydrate the silkfibroin solution and induce β-sheet structures in the silk fibroin; and(d) removing at least a portion of the lipids to produce a biomaterialthat has been encapsulated in silk fibroin microspheres.

Another embodiment of this invention relates to a silk fibroinmicrosphere composition, comprising a therapeutic agent encapsulated incrosslinked silk fibroin microspheres, wherein at least 75% of themicrospheres are spherical or substantially spherical, and wherein atleast 75% of the microspheres have a diameter ranging from 1.0 to 3.0μm.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts photomicrographs of particle suspensions obtained duringsilk microsphere preparation of: (A) DOPC film hydrated by water; (B)DOPC film hydrated by silk solution; (C) DOPC-silk mixture afterfreeze-thaw 3 times; and freeze-thawed and lyophilized DOPC-silksuspended in saturated NaCl solution at 10 min (D), 20 min (E), and 40min (F). The arrows indicate the fused lipid vesicles. Bar indicates 50μm.

FIG. 2 represents charts depicting the yield of microspheres when (A)silk and DOPC ratio were varied in MeOH-based silk microspheres, and (B)NaCl treatment time was varied in NaCl-based silk microspheres. Errorbars represent standard deviations from samples n=3.

FIG. 3 depicts SEM images of freeze-thawed and lyophilized DOPC-silksilk microspheres when untreated (A-D), treated with methanol (E-H), andtreated with NaCl for 15 h (I-L). Bar indicates 20 μm in A, E, I; 5 μmin B, F, J; and 2 μm in C, D, G, H, K, and L.

FIG. 4 depicts confocal laser scanning microscopy images of the silkmicrospheres containing fluorescein-labeled DOPE. Labeled phospholipidsremained in MeOH-based silk microspheres (A) and NaCl-based silkmicrospheres (B), forming either multilamellar structures (C) ornon-lamellar structure (D). Bar indicates 75 μm in A and B; 7.36 μm inC; and 10.77 μm in D.

FIG. 5 represents FTIR spectra (amide I band) of silk microspheresprepared (a) as lyophilized DOPC-silk suspended in water; (b), (c), and(e) as silk microspheres prepared with 1, 4, and 15 h NaCl treatment andsuspended in water, respectively; and (d) as silk microspheres preparedwith MeOH treatment.

FIG. 6 depicts confocal laser scanning microscopy images showing thedistribution of silk and drug in silk microspheres. Fluorescein-labeledsilk (left panels) and rhodamin B-labeled dextran 40,000 (middle panels)are located in separate layers (A) or domains (B) in MeOH-basedmicrospheres prepared from lyophilized DOPC, silk, and drug mixture. Thesame mixture when freeze-thawed prior to lyophilization shows that thesilk and drug are mixed in the same layers (C) and domains (D) in bothMeOH-based and NaCl-based microspheres. Images in the left and middlepanels are merged into the right panels. Bar indicates 5.29, 1.49, 2.24,and 3.67 μm in A, B, C and D, respectively.

FIG. 7 represents (A) the HRP release from MeOH-based silk microspheres() and DOPC-silk mixture prior to MeOH treatment (▪) and (B) the HRPrelease from NaCl-based silk microspheres after NaCl treatment for 1 h(▪), 4 h (), and 15 h (▴). Error bars represent standard deviationsfrom samples n=3.

FIG. 8 represents a schematic showing the process of preparingMeOH-based and NaCl-based microspheres.

DETAILED DESCRIPTION

This invention relates to a method of preparing silk fibroinmicrospheres. The method involves (a) mixing a silk fibroin solutionwith a lipid composition; (b) lyophilizing the mixture; (c) combiningthe lyophilized material with a dehydration medium for a sufficientperiod of time to at least partially dehydrate the silk fibroin solutionand induce β-sheet structures in the silk fibroin; and (d) removing atleast a portion of the lipids to form silk fibroin microspheres.

Silkworm fibroin is the structural protein of silk fibers. Silk fibroincan be fabricated easily into desired shapes, such as films,3-dimensional porous scaffolds, electrospun fibers, and hydrogels. Thesematerials have the advantage of excellent mechanical properties,biocompatibility and biodegradability. Silk fibroin solutions may beprepared as aqueous stock solution in accordance with the proceduresused by Sofia et al., “Functionalized silk-based biomaterials for boneformation,” J. Biomed Mater Res. 54 (2001) 139-148, herein incorporatedby reference in its entirety.

As used herein, the term “fibroin” includes silkworm fibroin and insector spider silk protein (Lucas et al., Adv. Protein Chem 13: 107-242(1958)). Preferably, fibroin is obtained from a solution containing adissolved silkworm silk or spider silk. The silkworm silk protein isobtained, for example, from Bombyx mori, and the spider silk is obtainedfrom Nephila clavipes. In the alternative, suitable silk proteins can beobtained from a solution containing a genetically engineered silk, suchas from bacteria, yeast, mammalian cells, transgenic animals ortransgenic plants. See, for example, WO 97/08315 and U.S. Pat. No.5,245,012.

In addition to the silk fibroin, the silk fibroin solution may alsocontain one or more therapeutic agents. The therapeutic agent may be anyagent known by those of skill in the art to have therapeutic properties.Suitable therapeutic agents include proteins, peptides (preferablytherapeutic peptides), nucleic acids, PNA, aptamers, antibodies, growthfactors, cytokines, enzymes, and small molecules (preferably smallmedicinal drug compounds having a molecular weight of less than 1000Da). Preferred therapeutic agents include morphogenetic protein 2(BMP-2), insulin-like growth factor I and II (IGF-I and II), epidermalgrowth factor (EGF), platelet-derived growth factor (PDGF), fibroblastgrowth factors (FGFs), transforming growth factors-β (TGFs-β),transforming growth factors-α, erythropoietin (EPO), interferon α and γ,interleukins, tumor necrosis factor α and β, insulin, antibiotics, andadenosine.

The therapeutic agent, when mixed with the silk fibroin solution, can beencapsulated in the silk fibroin microspheres. The encapsulatedtherapeutic agent can then be released from the microspheres throughtypical release mechanisms known in the art. Preferably, the therapeuticagent is in an active form when added to the silk fibroin and in anactive form when encapsulated in the silk fibroin microspheres. Keepingthe therapeutic agent in an active form throughout the microspherepreparation process enables it to be therapeutically effective uponrelease from the microsphere.

Biocompatible polymers can also be added to the silk fibroin solution togenerate composite matrices. Useful biocompatible polymers include, forexample, polyethylene oxide (PEO) (U.S. Pat. No. 6,302,848),polyethylene glycol (PEG) (U.S. Pat. No. 6,395,734), collagen (U.S. Pat.No. 6,127,143), fibronectin (U.S. Pat. No. 5,263,992), keratin (U.S.Pat. No. 6,379,690), polyaspartic acid (U.S. Pat. No. 5,015,476),polylysine (U.S. Pat. No. 4,806,355), alginate (U.S. Pat. No.6,372,244), chitosan (U.S. Pat. No. 6,310,188), chitin (U.S. Pat. No.5,093,489), hyaluronic acid (U.S. Pat. No. 6,387,413), pectin (U.S. Pat.No. 6,325,810), polycaprolactone (U.S. Pat. No. 6,337,198), polylacticacid (U.S. Pat. No. 6,267,776), polyglycolic acid (U.S. Pat. No.5,576,881), polyhydroxyalkanoates (U.S. Pat. No. 6,245,537), dextrans(U.S. Pat. No. 5,902,800), polyanhydrides (U.S. Pat. No. 5,270,419), andcombinations thereof (all parenthetical references are to U.S. patentnumbers, which illustrate an example of the referenced polymer).

Lipid vesicles are used in the process as templates to assist inmodeling the microspheres into preferred shapes and sizes. The lipidcomposition may include any lipid or combination of lipids that can formliposomes. Suitable lipids in the lipid composition include1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC);1,2-dioleoyl-sn-glycero-3-phophoethanolamine (DOPE);1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC); and1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Other lipidcompositions known in the art may also be used.

The silk fibroin solution and lipid composition should be mixed in amanner that integrates the silk fibroin and lipids. When a therapeuticagent is present in the silk fibroin solution, the therapeutic agent,silk fibroin, and lipids are all mixed together. Preferably, the mixingtakes place for a sufficient period of time and under conditions so thatthe various components are significantly integrated.

Sufficient mixing is sometimes difficult to achieve. In such cases, afreeze-thaw step may be used, which promotes mixing among the lipids,silk fibroin, and therapeutic agents, when present. A freeze-thaw stepcan break larger multilamellar lipid vesicles into smaller, unilamellarstructures that have more homogeneous size distributions. It can also beused to facilitate silk self assembly and enhance the encapsulation ofthe therapeutic agent in the liposomes.

Any freeze-thaw treatment known in the art may by used. See, e.g.,Colletier et al., “Protein encapsulation in liposomes: efficiencydepends on interactions between protein and phospholipid bilayer,” BMCBiotechnology 2 (2002) 9-17, herein incorporated by reference in itsentirety, for suitable freeze-thaw techniques. The freeze-thaw may berepeated one or more times to promote further mixing and sizehomogeneity. Freeze-thawing is not deemed necessary when using certaindehydrating mediums, such as methanol, where the particle-sizedistribution and integration level achieved through mixing alone isusually adequate.

The amount of silk fibroin solution and lipid composition that is mixedis dependent on the dehydrating medium used and the desired structuralformation of the microspheres. Typically, 0.1 to 2 ml of 8 (w/v) % silksolution is used for every 100 mg of lipids. However, these amounts mayvary depending on the exact make up of the silk solution and lipidcomposition. Additionally, depending on the dehydrating medium used,each medium will have a threshold ratio. For instance, when methanol isused as the dehydrating medium, the threshold ratio is 0.2 ml of 8 (w/v)% silk solution for every 100 mg of lipids, and when sodium chloride isused as the dehydrating medium, the threshold ratio is 0.5 ml of 8 (w/v)% silk solution for every 100 mg of lipids. When the amount of lipidsare above the threshold ratio, multilamellar structures predominatelyform in the microspheres; when the amount of lipids are below thethreshold ratio, unilamellar structures predominately form in themicrospheres.

The lipid components that remain in the microspheres will form as eitheruni- or multilamellar structures. Compared to multilamellar lipidvesicles, unilamellar vesicles offer higher encapsulation capacity forhydrophilic drugs, more reproducible rates of release, and less lipidcontent in the microspheres. On the other hand, multilamellar vesiclesare suitable for encapsulating both lipophilic and hydrophilic drugs andare more resistant to enzyme digestion, resulting in a longercirculation time in the body. Therefore, unilamellar-structuredmicrospheres are generally preferred when higher drug loading is neededor when hydrophilic drugs are used; multilamellar-structuredmicrospheres are generally preferred when lipophilic drugs are used andin cases when drug loading is not important or when a slower degradationof microspheres is desired. In addition to vesicle structure (uni- ormultilamellar), the drug release rate is also governed by lipophilicityof drug molecules, the composition of the encapsulation device, and thelipid composition.

After the silk fibroin solution and the lipid composition have beenmixed and optionally freeze-thawed, the mixture is lyophilized.Lyophilization techniques known in the art may be used. Typically, themixture is lyophilized for three days and stored at temperatures around4° C.

The lyophilized material is then combined with a dehydration medium. Thedehydration medium may be any medium that can both dehydrate the silkfibroin solution and induce β-sheet structures in the silk fibroin.Dehydrating the silk fibroin solution extracts water from the silkfibroin and causes the silk to self assemble and form crystallineβ-sheet structures. The β-sheet structures are physical crosslinks inthe silk fibroin that provide the silk with stability and uniquemechanical features in the fibers. The physical crosslinks also promotethe entrapment of therapeutic agents, when present, in the silk fibroin.Beta-sheet structures in the silk fibroin may also be induced by changesin salt concentration and shear forces.

Microspheres will form upon crosslinking of the silk fibroin.Preferably, the weight percentage of microspheres in the total silk isat least about 50%. The amount of microspheres in the silk is dependenton various factors, such as the dehydration agent used to induce β-sheetstructure, the type of silk fibroin used, the amount of time the silk isexposed to the dehydration agent, etc. If a therapeutic agent wasintroduced in the process, then the silk fibroin microspheres canencapsulate the therapeutic agent during microsphere formation.

The dehydration medium should at least partially dehydrate the silkfibroin. Preferably, the silk fibroin is sufficiently dehydrated so thatsignificant amounts (e.g. 50% or more) of β-sheet structures form in thesilk. The amount of dehydration time necessary to induce β-sheetformation is readily determinable by one skilled in the art and willdepend, in part, on the dehydration medium used. Because highcrystallinity can significantly retard the release of encapsulatedtherapeutic agents, such as proteins, inducing large amounts of β-sheetformation is preferable when forming microspheres designed for controlrelease.

Any known dehydration medium that does not destroy or otherwise damagethe silk fibroin may be used as the dehydration medium. Polar alcohols,such as methanol and ethanol, are particularly effective at inducingdehydration of the silk. Other polar solvents, such as acetone, are alsoeffective. Solvents and alcohols with lower polarity, such as chloroformand propanol, may also be used, but are not as effective at stabilizingthe silk structure. Additionally, many salts, such as sodium chlorideand potassium chloride, can dehydrate the silk fibroin as well changethe salt concentration, both of which induce β-sheet formation. Othersuitable dehydration mediums include polyethylene glycol solutions,desiccants, and dry gas. Preferably, the dehydration medium is a polarsolvent, such as methanol, ethanol, and acetone, or a salt, such assodium chloride or potassium chloride. Methanol and solutions of sodiumchloride are particularly preferred.

The lyophilized material and dehydration medium may be combined throughany method known in the art. Preferably, the dehydration medium is in asolution and the lyophilized material is combined with it by adding thelyophilized material to the solution containing the dehydration medium.Combining the two components in this manner will typically form asuspension of the lyophilized material in the dehydration mediumsolution. When the lyophilized material is suspended in the solution, itallows for easier removal of the lipids.

At least some of the lipids should be removed after the lyophilizedmaterial has been combined with the dehydration medium. The lipids maybe removed through any technique known in the art. Centrifugation may beused when the lyophilized material is suspended in a solution containingthe dehydration medium, however, other removal or extraction techniquesmay be better suited to remove the lipids depending on the dehydrationmedium utilized.

Certain dehydration mediums can function to remove the lipids. Forinstance, a high concentration of methanol or sodium chloride enableseach medium to function as both a dehydration medium and lipid remover.Additional removal steps, such as centrifugation, are nonetheless stillpreferred even when using methanol or sodium chloride. Other dehydrationmediums, such as desiccants or dry gas, function little if at all as alipid remover. These type of dehydration mediums, therefore, may have tobe combined with a more rigorous lipid extraction or removal step, ormultiple extraction/removal steps.

It is preferable to remove all or substantially all of the removablelipids. Depending on the removal techniques and dehydration medium used,complete lipid removal may not be possible. For instance, when usingmethanol as the dehydration medium, about 99% of the lipids are able tobe removed; when using sodium chloride as the dehydration medium, about83% of the lipids are able to be removed. In these cases, all orsubstantially all of the removable lipids are considered to have beenremoved because further removal techniques would not lead to anysubstantial amount of additional lipids being removed.

While it is preferable to remove most of the lipids, it is also believedthat the lipid components, when present in a relatively small amount,can be beneficial. In particular, it is believed that the lipidcomponent can assist in controlling the release of the therapeutic agentfrom the microspheres. Therefore, according to an embodiment of theinvention, it is preferable to have a microsphere composition whereabout 15 to about 20% of the total lipids remain in the silk fibroinmicrospheres. It is also preferable to have a microsphere compositionwhere less than about 5% of the total lipids remain in the silk fibroinmicrospheres. More preferably, less than about 2% of the total lipidsremain in the microspheres.

After the desired amount of lipids have been removed, the composition istypically in a dehydrated pellet form. The composition may be hydratedby suspending or resuspending the microsphere composition in water or abuffer solution. Suspending the microspheres in water or a buffer isoften done before the microsphere composition is used in a commerciallyviable manner. For instance, if the silk fibroin microspheres are usedin a formulation suitable for administration, the formulation willtypically contain hydrated microspheres.

A pharmaceutical formulation may be prepared that contains the silkfibroin microspheres having encapsulated therapeutic agents. Theformulation can be administered to a patient in need of the particulartherapeutic agent that has been encapsulated in the microspheres.

The pharmaceutical formulation may be administered by a variety ofroutes known in the art including topical, oral, parenteral (includingintravenous, intraperitoneal, intramuscular and subcutaneous injectionas well as intranasal or inhalation administration) and implantation.The delivery may be systemic, regional, or local. Additionally, thedelivery may be intrathecal, e.g., for CNS delivery.

In addition to the silk microspheres, the pharmaceutical formulation mayalso contain a targeting ligand. Targeting ligand refers to any materialor substance which may promote targeting of the pharmaceuticalformulation to tissues and/or receptors in vivo and/or in vitro with theformulations of the present invention. The targeting ligand may besynthetic, semi-synthetic, or naturally-occurring. Materials orsubstances which may serve as targeting ligands include, for example,proteins, including antibodies, antibody fragments, hormones, hormoneanalogues, glycoproteins and lectins, peptides, polypeptides, aminoacids, sugars, saccharides, including monosaccharides andpolysaccharides, carbohydrates, vitamins, steroids, steroid analogs,hormones, cofactors, and genetic material, including nucleosides,nucleotides, nucleotide acid constructs, peptide nucleic acids (PNA),aptamers, and polynucleotides. Other targeting ligands in the presentinvention include cell adhesion molecules (CAM), among which are, forexample, cytokines, integrins, cadherins, immunoglobulins and selectin.

The pharmaceutical formulations may also encompass precursor targetingligands. A precursor to a targeting ligand refers to any material orsubstance which may be converted to a targeting ligand. Such conversionmay involve, for example, anchoring a precursor to a targeting ligand.Exemplary targeting precursor moieties include maleimide groups,disulfide groups, such as ortho-pyridyl disulfide, vinylsulfone groups,azide groups, and iodo acetyl groups.

The pharmaceutical formulations may contain common components found inother pharmaceutical formulations, such as known excipients. Exemplaryexcipients include diluents, solvents, buffers, solubilizers, suspendingagents, viscosity controlling agents, binders, lubricants, surfactants,preservatives and stabilizers. The formulations may also include bulkingagents, chelating agents, and antioxidants. Where parenteralformulations are used, the formulation may additionally or alternatelyinclude sugars, amino acids, or electrolytes.

Suitable excipients include polyols, for example, of a molecular weightless than about 70,000 kD, such as trehalose, mannitol, and polyethyleneglycol. See for example, U.S. Pat. No. 5,589,167, the disclosure ofwhich is incorporated by reference herein. Exemplary surfactants includenonionic surfactants, such as Tweeng surfactants, polysorbates, such aspolysorbate 20 or 80, etc., and the poloxamers, such as poloxamer 184 or188, Pluronic polyols, and other ethylene/polypropylene block polymers,etc. Suitable buffers include Tris, citrate, succinate, acetate, orhistidine buffers. Suitable preservatives include phenol, benzylalcohol, metacresol, methyl paraben, propyl paraben, benzalconiumchloride, and benzethonium chloride. Other additives includecarboxymethylcellulose, dextran, and gelatin. Suitable stabilizingagents include heparin, pentosan polysulfate and other heparinoids, anddivalent cations such as magnesium and zinc.

The pharmaceutical formulations containing the microspheres can beadministered in a controlled-release manner so that portions of thetherapeutic agent are released in the patient over a period of time. Thetherapeutic agent may release quickly or slowly. For instance, thepharmaceutical formulation can be administered so that less than about5% of the therapeutic agent is released in the patient from themicrospheres over a period of one month. Alternatively, a larger portionof the therapeutic agent may be released initially, with a smallerportion retained in the microspheres and released later. For example,the pharmaceutical formulation can be administered so that at least 5%of the therapeutic agent remains in the microspheres 10 days afteradministration.

When administering the therapeutic agent in a controlled-release manner,the therapeutic agent preferably remains active in the microspheres sothat it can perform its therapeutic function upon release. Certaintherapeutic agents become inactive when exposed to encapsulationconditions for a significant period time. Of course, the release ofinactive therapeutic agents is of little or no value to the patient, whois not able to receive the benefits of an active therapeutic agent. Apreferred pharmaceutical formulation contains microspheres where theactivity of the therapeutic agent in the microspheres remains at atleast 50% one month after administration to the patient.

Controlled release permits dosages to be administered over time, withcontrolled release kinetics. In some instances, delivery of thetherapeutic agent is continuous to the site where treatment is needed,for example, over several weeks. Controlled release over time, forexample, over several days or weeks, or longer, permits continuousdelivery of the therapeutic agent to obtain preferred treatments. Thecontrolled delivery vehicle is advantageous because it protects thetherapeutic agent from degradation in vivo in body fluids and tissue,for example, by proteases.

Controlled release from the pharmaceutical formulation may be designedto occur over time, for example, for greater than about 12 or 24 hours.The time of release may be selected, for example, to occur over a timeperiod of about 12 hours to 24 hours; about 12 hours to 42 hours; or,e.g., about 12 to 72 hours. In another embodiment, release may occur forexample on the order of about 2 to 90 days, for example, about 3 to 60days. In one embodiment, the therapeutic agent is delivered locally overa time period of about 7-21 days, or about 3 to 10 days. In otherinstances, the therapeutic agent is administered over 1, 2, 3 or moreweeks in a controlled dosage. The controlled release time may beselected based on the condition treated. For example, longer times maybe more effective for wound healing, whereas shorter delivery times maybe more useful for some cardiovascular applications.

Another embodiment of this invention relates to a drug deliverycomposition comprising a therapeutic agent encapsulated in crosslinkedsilk fibroin microspheres, wherein the microspheres contain lipidcomponents. The silk fibroin microspheres may be crosslinked by exposingthe silk fibroin to a dehydrating medium, such as methanol or sodiumchloride, which induces β-sheet formation, or the crosslinking of thesilk fibroin.

When silk fibroin microspheres are prepared with a process that utilizeslipid components, a portion of the lipid components is typically presentin the silk fibroin microspheres, even when all of the removable lipidcomponents have been removed. Depending on the process used toincorporate and/or remover the lipids, lipid components will typicallybe present in the microspheres from about 1 to about 25%, by weight.Preferably, the microspheres contain less than about 20% lipids byweight, more preferably less than about 5% lipids by weight. It isbelieved that the lipids, when present in relatively small amounts,assist in controlling the release of the therapeutic agent from themicrospheres. When the microspheres contain too high a percentage oflipids, the structure and physical parameters of the silk fibroinmicrospheres can be compromised, resulting in less effectivemicrospheres or microspheres with insufficient structural integrity.

Another embodiment of this invention relates to a method ofencapsulating a biomaterial in silk fibroin microcapsules. The methodcomprises (a) mixing a solution comprising silk fibroin and abiomaterial with a lipid composition; (b) lyophilizing the mixture; (c)combining the lyophilized material with a dehydration medium for asufficient period of time to at least partially dehydrate the silkfibroin solution and induce β-sheet structures in the silk fibroin; and(d) removing at least a portion of the lipids to produce a biomaterialthat has been encapsulated in silk fibroin microspheres.

The biomaterial may be a therapeutic agent, such one or more of thetherapeutic agents discussed above. However, the encapsulation processdoes not have to be used in the field of pharmaceutical formulations andcontrolled-release methods. The silk fibroin microcapsules mayencapsulate various other biomaterials useful in a variety of fields.For instance, the biomaterial may be an enzyme or an enzyme-basedelectrode. The enzyme or enzyme-based electrode may be used in the fieldof tissue engineering, biosensors, the food industry, environmentalcontrol, or biomedical applications. The system can also be used as areservoir for a variety of needs, such as in the food industry to harborvitamins, nutrients, antioxidants and other additives; in theenvironmental field to harbor microorganisms for remediation or watertreatments; in materials as additives to serve as a source of in situdetection and repair components, such as for nondestructive evaluationof material failures and self-repairs of the materials; and forbiodetection schemes to help stabilize cells, molecules and relatedsystems.

The silk fibroin microspheres of the invention form in a manner thatprovides them with advantageous physical properties that areparticularly useful for encapsulating therapeutic agents for uses incontrolled-release pharmaceutical formulations. The microspheres exhibita more homogeneous shape and size, especially when compared tomicrospheres prepared via conventional techniques, such as spray-drymethods. Exhibiting a homogeneous spherical shape, the microspheres areless likely to experience aggregation, which occurs more commonly whenthe microspheres are in a funicular (fibrillar or elongated) state. Thesmaller and more narrow diameter range of microspheres also provides amore consistent and controlled release.

Accordingly, an embodiment of this invention relates to a silk fibroinmicrosphere composition, comprising a therapeutic agent encapsulated incrosslinked silk fibroin microspheres, wherein at least 75% of themicrospheres are spherical or substantially spherical, and wherein atleast 75% of the microspheres have a diameter ranging from about 1.0 toabout 3.0 μm. Preferably, at least 90% of the microspheres are sphericalor substantially spherical, and at least 90% of the microspheres have adiameter ranging from about 1.0 to about 3.0 μm. More preferably, atleast 95% of the microspheres have a diameter ranging from about 1.0 toabout 3.0 μm. The average size of the microspheres is preferably lessthan about 2.0 μm. The silk microspheres with small sizes are of moreinterest for biomedical applications

The size and shape of the microsphere will be dependent, to some degree,on what techniques are used to crosslink the silk fibroin. For instance,dehydrating the silk fibroin in methanol in the above-described methodswill typically produce microspheres wherein about 90% of themicrospheres are substantially spherical and about 90% have a diameterranging from 1.0 to 3.0 μm. The term “substantially spherical,” as usedherein, means spherical microspheres that contain small blemishes in thesurface or on the edges of the microspheres, but that would otherwise beconsidered spherical as opposed to funicular. See FIG. 3E, depictingsubstantially spherical microspheres. Using these methods with sodiumchloride will typically produce microspheres wherein about 90% of themicrospheres are spherical and about 98% have a diameter ranging from1.0 to 3.0 μm. See FIG. 3I, depicting spherical microspheres.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of theinvention, the preferred methods and materials are described below. Allpublications, patent applications, patents and other referencesmentioned herein are incorporated by reference. In addition, thematerials, methods and examples are illustrative only and not intendedto be limiting. In case of conflict, the present specification,including definitions, controls.

The invention will be further characterized by the following exampleswhich are intended to be exemplary of the invention.

EXAMPLE Materials

Cocoons of B. mori silkworm silk were supplied by M. Tsukada (Instituteof Sericulture, Tsukuba, Japan).1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein)(fluorescein-DOPE) were purchased from Avanti Polar Lipids (Alabaster,Ala.). 5-(Aminoacetamido)fluorescein (fluoresceinyl glycine amide) waspurchased from Molecular Probes (Carlsbad, Calif.). Rhodamine βisothiocyanate-Dextran (M.W. 40,000 Da), horseradish peroxidase (HRP),β-galactosidase, and other chemicals were obtained from Sigma Aldrich(St. Louis, Mo.). 3,3′5,5′ Tetramethylbenzidine (TMB) solution waspurchased from BioFX laboratories (Owing Mills, Md.).1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC),N-hydroxysuccinimide (NHS), and hydroxylamine hydrochloride werepurchased from Pierce Biotechnology (Rockford, Ill.). All otherchemicals were purchased from Sigma-Aldrich (St. Louis, Mo.).

Purification and Fluorescent Labeling of Silk Fibroin:

Silk fibroin aqueous stock solutions were prepared as described byBrandl, “Liposomes as drug carriers: a technological approach,”Biotechnol. Ann. Rev. 7 (2001) 59-85, herein incorporated by referencein its entirety. Briefly, cocoons of B. mori were boiled for 20 min inan aqueous solution of 0.02 M sodium carbonate, and then rinsedthoroughly with pure water. After drying, the extracted silk fibroin wasdissolved in 9.3 M LiBr solution at 60° C. for 4 hours, yielding a 20%(w/v) solution. This solution was dialyzed against distilled water usingSlide-a-Lyzer dialysis cassettes (MWCO 3,500, Pierce) for 3 days toremove the salt. The solution was clear after dialysis and wascentrifuged to remove silk aggregates (small amount) that formed duringthe dialysis and some dirt from cocoons. The final concentration of silkfibroin aqueous solution was approximately 8% (w/v). This concentrationwas determined by weighing the residual solid of a known volume ofsolution after drying.

For fluorescent labeling, the silk fibroin stock solution was diluted to2% (w/v) with water, and 10 ml of the diluted solution was dialyzedagainst 500 ml of 0.1 M 2-(morpholino)ethanesulfonic acid (MES) solution(pH 5.6) (Pierce, Chemicals, IL) supplemented with 0.9% NaCl overnight.Eighty mg EDC (2 mM) and 220 mg NHS (5 mM) were added to the bufferedsilk solution with stirring and the reaction was continued for 15 min.β-mercaptoethanol was added to a final concentration of 20 mM to quenchthe unreacted EDC. The carboxyl groups on silk fibroin were activatedfor reacting with primary amines. After the reaction, 10 mg offluoresceinyl glycine amide was added to the solution so that the molarratio between fluorescent probe and silk fibroin was about 40:1. Thecoupling reaction went for 2 hours under slow stirring at roomtemperature and then 8 mg hydroxylamine hydrochloride was added toquench the reaction. Finally the solution was dialyzed exhaustivelyagainst water. The final concentration of fluorescent silk fibroin wasapproximately 1.5% (w/v) using the same weighing method.

Preparation of Silk Microspheres:

One hundred mg of DOPC was dissolved in 1 ml chloroform in a glass tubeand dried into a film under a flow of nitrogen gas. 8% (w/v) silkfibroin solution with volume of 0.33 ml, 0.5 ml, and 1 ml was added tohydrate the lipid film, and the mixture was diluted to 2 ml with waterand moved to a plastic tube. The sample was frozen in liquid nitrogenfor 15 min and then thawed at 37° C. for 15 min. This freeze-thaw cyclewas repeated 3 times and then the thawed solution was slowly pipettedinto a glass beaker containing 50 ml water with fast stirring. Formethanol-treated microspheres, the freeze-thaw treatment was skipped andthe 0.5 ml of DOPC-silk mixture was diluted to 50 ml water directly. Theresulting solution was lyophilized for 3 days and stored at 4° C.

To prepare MeOH— based microspheres, 20 mg lyophilized material wassuspended in 2 ml MeOH in an Eppendorf tube and the suspension wasincubated for 30 min at room temperature followed by centrifugation at10,000 rpm for 5 min at 4° C. (Eppendorf 5417R centrifuge). The pelletobtained was dried in air and stored at 4° C. To generate a suspensionof silk microspheres, the dried pellet was washed once with 2 ml ofwater by centrifugation, and then resuspended in the desired water orbuffer. The clustered microspheres were dispersed by ultrasonication for10 sec at 30% amplitude (approximately 20 W) using a Branson 450ultrasonicator (Branson Ultrasonics Co., Danbury, Conn.).

To prepare NaCl-based microspheres, 20 mg lyophilized material wassuspended in 2 ml saturated NaCl solution in an Eppendorf tube and thesuspension was incubated at room temperature for 1 h, 4 h, and 15 hfollowed by centrifugation at 10,000 rpm for 5 min at 4° C. (Eppendorf5417R centrifuge). The supernatant and the white viscous materialfloating on the top were carefully removed, and the pellet was washedonce with 2 ml water by centrifugation and then resuspended in water orbuffer.

Phospholipids Quantification

Phospholipids remained in the silk microspheres and were estimated byphosphorus determination through an acidic digestion. See Rouser et al.,“Two dimensional then layer chromatographic separation of polar lipidsand determination of phospholipids by phosphorus analysis of spots,”Lipids 5 (1970):494-496, and Zhou et al., “Improved procedures for thedetermination of lipid phosphorus by malachite green,” J. Lipid Res. 33(1992):1233-1236, both of which are herein incorporated by reference intheir entirety. The released phosphorus was reacted with ammoniummolybdate to form a strong blue color.

Dried MeOH-based and NaCl-based silk microspheres were weighed andtransferred into clean glass tubes. 0.65 ml perchloric acid was added toeach sample, and the tubes were heated at 180° C. until the yellow colorin all the tubes disappeared. When cool, the tubes were supplementedwith 3.3 ml water, 0.5 ml 2.5% (w/v) molybdate solution and 0.5 ml 10%(w/v) ascorbic acid solution. The tubes were agitated on a vortex aftereach addition. The samples were then boiled in a water bath for 5 min,and the absorbance of cool samples (including the standards) was read at800 nm. Potassium phosphate monobasic (KH₂PO₄) solution was used as astandard. The stock solution of 439 mg per liter water (i.e., 100 μgphosphorus per milliliter water) was diluted in 3.3 ml water and 0.65 mlperchloric acid. Digestion at 180° C. was not necessary before addingreagents. The amount of phospholipids was calculated directly on aweight basis after multiplying the amount of phosphorus by 25.38 (DOPCcontains 3.94% w/w phosphorus).

Dynamic Light Scattering (DLS)

Microspheres were diluted in 10 ml water in a glass vial and analyzedimmediately at 25° C. using a BIC BI-200 SM research goniometer andlaser light scattering system (Brookhaven Instrument, Holtsville, N.Y.).Laser light at 532 nm was used to measure the fluctuation in intensityof light scattered by particles. Data were collected for 5 min for eachsample, and the mean diameter of particles was calculated using the BICdynamic light scattering software supplied by the manufacturer of theabove-referenced system.

Fourier Transform Infrared (FTIR) Spectroscopy

FTIR studies were performed using a Bruker Equinox 55 FTIR spectrometer.A drop of microsphere suspension was added to the zinc selenide (ZnSe)crystal cell and examined with the FTIR microscope in the reflectionmode. Background measurements were taken with an empty cell andsubtracted from the sample reading. DOPC suspended in water did not showpeaks at the amide I band region, meaning that its influence wasnegligible. Deconvolution of the fibroin amide I spectra was performedusing Gaussian×Lorentzian function in the spectroscopic software fromBriler (version 4.2). The curves that had absorption bands at thefrequency range of 1620-1630 cm⁻¹ and 1695-1700 cm⁻¹ representedenriched β-sheet structure in silk II form (23). The contribution ofthese curves (β-sheet structure content) to the amide I band wasassessed by integrating the area under the curve and then normalizing tothe total area under the amide I band region (1600-1700 cm⁻¹).

Scanning Electron Microscopy (SEM)

For lyophilized DOPC-silk and MeOH-based microspheres, dried materialswere directly mounted on samples mounts. For NaCl-based microspheres,the solution containing microspheres were dried on plastic slides whichwere further cut and mounted. Specimens were then sputter-coated with Auusing a Ploaron SC502 Sputter Coater (Fison Instruments, UK), andexamined using a JEOL JSM 840A Scanning Electron Microscope (Peabody,Mass.) at 15 KV.

Phase Contrast and Confocal Laser Scanning Microscopy

Microspheres were suspended in pure water and approximately 20 μl ofsuspension was put on a glass slide and covered with a cover-slip. Thesamples were analyzed by a phase contrast light microscope (Carl Zeiss,Jena, Germany) equipped with a Sony Exwave HAD 3CCD color video camera,or a confocal laser scanning microscope (TCS Leica SP2, Welzlar,Germany) with Leica Confocal Software, version 2.5 (Leica Microsystems,Heidelberg, Germany).

HRP Loading and Release

Silk microspheres were prepared as described above except that 10 μl ofrhodamine B-labeled dextran, 40,000 Da or HRP stock solution at 12.5mg/ml in buffer, were mixed with 0.5 ml of 8% (w/v) silk solution priorto microsphere formation. Dulbecco's phosphate buffer, pH 7.2(Invitrogen, Carlsbad, Calif.) was used for all HRP determinations. Forloading and release, 40 mg of lyophilized DOPC-silk fibroin was treatedwith MeOH or NaCl as described. After washing with buffer, themicrospheres were suspended in 2 ml of phosphate buffer, pH 7.2. One mlaliquots of the suspension were used for HRP loading, and the other 1 mlaliquot for HRP release. TMB (HRP substrate, Mw=240 Da) was oxidizedduring the enzymatic degradation of H₂O₂ by HRP. The oxidized product ofTMB exhibited a deep blue color which turned to yellow upon addition ofthe acidic stop solution.

For loading determinations, 5 μl of the suspension was mixed with 100 μlof TMB solution in 96-well standard microplate wells for 1 min at roomtemperature. The reaction was stopped by the addition of 100 μl 0.1 Msulfuric acid. Absorbance was detected at 450 nm by using a VersaMaxmicroplate reader (Molecular devices, Sunnyvale, Calif.). The HRPcontent was obtained using a HRP standard curve generated under the samecondition. The remaining microspheres (995 μl) were spun down, dried andweighed. The loading was obtained as follows:

${{Loading}( {\mu \; g\text{/}{mg}} )} = \frac{{HRP}\mspace{14mu} {{content}( {\mu \; g} )} \times 199}{{Weight}\mspace{14mu} {of}\mspace{14mu} {{microspheres}({mg})}}$

The loading efficiency was calculated as follows:

${{Loading}\mspace{14mu} {{efficiency}(\%)}} = \frac{{HRP}\mspace{14mu} {{loading}( {\mu \; g\text{/}{mg}} )} \times {Total}\mspace{14mu} {{microspheres}({mg})}}{{Total}\mspace{14mu} {{HRP}( {\mu \; g} )}}$

To determine HRP release, 1 ml suspensions of silk microspheres wereincubated at room temperature. At desired time points, the suspensionswere centrifuged at 10,000 rpm for 2 min. The supernatant was carefullymoved to another tube and the pellet was resuspended in 1 ml freshbuffer. HRP content in the supernatant was determined as described aboveand the percentage of release was obtained by comparing this data withthe loading data. All experiments were performed in triplicate.Statistical analysis of data was performed using the Student's t-test.Differences were considered significant when p<0.05.

Liposome-Assisted Silk Microsphere Preparation

FIG. 1 shows the microscopic images of particle suspensions that weregenerated in the different steps. As a control, hydration of the DOPCfilm with water resulted in highly dispersed vesicles with aheterogeneous size distribution (FIG. 1A). Hydration of DOPC films withsilk fibroin solution resulted in clustered vesicles with similarheterogeneous size distributions (FIG. 1B). Once the DOPC-silk mixturewas freeze-thawed 3 times, the water suspension was dominated by highlydispersed particles with a homogeneous size distribution (FIG. 1C). Oncethe freeze-thawed and lyophilized DOPC-silk was suspended in saturatedNaCl solution, some particles fused in time into larger lipid vesicles(FIGS. 1D-E). During preparation, these larger lipid vesicles floated ontop of the NaCl solution and could be removed by subsequentcentrifugation. The reason that some vesicles tend to fuse in this caseis probably due to high lipid content within the vesicles. Those withlow lipid but high silk content could survive and be treated into solidNaCl-based microspheres that were precipitated by centrifugation.Similarly, MeOH dissolved those lipid-rich vesicles but treated thosesilk-rich vesicles into MeOH-based microspheres.

Yield of Silk Microspheres

The lipid-to-silk ratio was adjusted to obtain a high yield ofmicrospheres (the weight percentage of microspheres in the total silk).In this example, MeOH treatment was used, and the weights ofmicrospheres were compared with the total silk that was originallyadded. As shown in FIG. 2A, a yield of about 55% was obtained when 40 mgof silk (0.5 ml 8% w/v silk solution) was mixed with 100 mg DOPC. Silkwas encapsulated to a saturated level in the lipid vesicles at thisratio but it was diluted when below the ratio and, therefore, was easierto be dispersed by MeOH. Thus, 0.5 ml 8% w/v silk solution and 100 mgDOPC was used as a standard condition for other preparations.Thirty-minute treatment time was used to prepare MeOH-basedmicrospheres, which was found to be sufficient to induce characteristicsilk II β-sheet structures. For NaCl-based preparations, the yields werereported in FIG. 2B. The yield of microspheres was significantlyincreased with NaCl treatment time, indicating that long NaCl treatmenttime (at least 15 h) is preferred for lipid removal and silkself-assembly, which is consistent with the observation by microscopicstudy (FIG. 1) and FTIR study (FIG. 3).

Particle Sizes

MeOH-based microspheres had an average size of 1.7 μm, as determined bydynamic light scattering (Table 1). The average size of NaCl-basedmicrospheres decreased with time of NaCl treatment, from 2.7 μm for 1hour to 1.6 μm when treated for 15 hours (Table 1), indicating that thesilk microspheres became more condensed upon NaCl-treatment.

As shown in FIGS. 3 E-H, approximately 90% of the methanol-basedmicrospheres have a particle size ranging from 1.0 μm to 3.0 μm. Asshown in FIGS. 3 I-L, approximately 98% of the sodium chloride-basedmicrospheres have a particle size ranging from 1.0 μm to 3.0 μm.

Phospholipid Content

The phospholipids contents remained in the silk microspheres weredetermined by phosphorus assay as described in the materials andmethods. The result showed that the MeOH-based and NaCl-basedmicrospheres contained about 1% w/w and 17% w/w DOPC, respectively(Table 1).

TABLE 1 Characteristics of silk microspheres DOPC-silk DOPC-silkMeOH-based 1 h NaCl 4 h NaCl 15 h NaCl DOPC-silk MeOH Freeze-thaw MS MSMS MS Particle size (μm)¹ — — —  1.73 ± 0.11 2.70 ± 0.35 2.24 ± 0.171.60 ± 0.09 (mean ± SD) n = 3 Phospholipids content² — — — 0.965 ± 0.16— — 17.13 ± 2.14  (%) HRP loading³ 0.082 ± 0.006 0.086 ± 0.013 0.165 ±0.012 0.173 ± 0.02 0.062 ± 0.007 0.109 ± 0.013 0.148 ± 0.019 (ug/mg silkMS) HRP Loading 9.8 ± 0.7 9.6 ± 1.4 19.8 ± 1.4 20.8 ± 2.4 7.4 ± 0.8 13.1± 1.6  17.8 ± 2.2  efficiciency⁴ (%) ¹Determined by dynamic lighterscattering. Standard deviation (SD) obtained based on threemeasurements. ²Phospholipids content represents the weight percentage ofphospholipids in microspheres. ³Determined by directly mixing substrateTMB with the microspheres suspended in buffer. ⁴Calculated by comparingthe amount of HRP determined in the silk microspheres with the totalamount of HRP added at the beginning.

Surface Morphology

The lyophilized DOPC-silk microspheres showed a smooth surface by SEM(FIG. 3, A-D). A similar surface morphology was observed for theNaCl-treated microspheres (FIG. 3, I-L). The MeOH-based microspheresexhibited a rougher surface that displayed minor defects at thesub-micron level (FIG. 3, E-H). It is believed that the difference insurface morphology between MeOH— and NaCl-based microspheres might havereflected their difference in phospholipids contents.

As shown in FIGS. 3 E-H, approximately 90% of the methanol-basedmicrospheres have a substantially spherical shape. As shown in FIGS. 3I-L, approximately 90% of the sodium chloride-based microspheres have aspherical shape.

Lamellar Structures

Fluorescent probe (fluorescein)-labeled DOPE was used to trace thephospholipids remaining in the microspheres using confocal laserscanning microscopy. Phospholipids remained in the MeOH— and NaCl-basedsilk microspheres (FIGS. 4A and B), forming either multilamellar (FIG.4C) or unilamellar structures (FIG. 4D). The formation of lamellarstructure is believed to be influenced by the ratio between lipid andsilk in a microsphere: Once the ratio is above a critical level, lipidwill dominate the formation of multilamellar structures, while belowthis level the silk fibroin would dominate the formation of unilamellarstructures.

Silk β-Sheet Structures

The β-sheet content in the MeOH— and NaCl-based microspheres wasassessed by FTIR (FIG. 5). When the NaCl treatment time was increased,the absorbance at the region of random coil, α-helix, and turn and bend(1640-1690 cm⁻¹) significantly decreased (curve b, c, e in FIG. 5),indicating that the β-sheet structure (silk II band at characteristicregion (1620-1630 cm⁻¹)) was increasing. Deconvolution of the curvesshowed that the initial material, freeze-thawed and lyophilizedDOPC-silk, contained about 29% β-sheet structure, which is slightlyhigher than the 25% content that has been reported for soluble silkfibroin in an aqueous solution. This indicates that the proteinstructure was not significantly influenced by the freeze-thaw andlyophilization process under the experimental condition (mixed withlipids). The NaCl-based microspheres with 1 h, 4 h, and 15 h NaCltreatment showed β-sheet contents of about 34%, 51%, and 67%,respectively. MeOH-based microspheres also showed high β-sheet contentof about 58%. These trends indicate that the β-sheet content in silkmicrospheres increases as the micropshere size decreases.

Controlled Drug Release 1. Silk and Drug Distribution in SilkMicrospheres

The distribution of fluorescein-labeled silk (green) and rhodamine Blabeled dextran 40,000 (red) in microspheres was studied by confocallaser scanning microscopy. When the freeze-thaw step was not included inthe preparation, silk and dextran were found to locate in separatelayers (FIG. 6A) or domains (FIG. 6B) in the MeOH-based microspheres.Once the freeze-thaw treatment was performed before lyophilization, inboth MeOH— and NaCl-based microspheres, the silk and dextran were mixedin the layers (FIG. 6 C, D). Freeze-thaw was used to promote mixingbetween the silk fibroin and the rhodamine B-labeled dextran 40,000.

2. HRP Loading in Silk Microspheres

Loading was determined in lyophilized DOPC-silk with and withoutfreeze-thaw. The freeze-thaw step increased the loading and loadingefficiency by approximately two-fold when compared to thenon-freeze-thawed samples (first and third columns in Table 1). Thismight be because the freeze-thaw treatment helped mix silk and drug inthe microspheres so that more drug molecules could be packed into themicrospheres. MeOH treatment on both samples did not deactivate the HRPand, therefore, the loading and loading efficiency were not changed inthe corresponding MeOH-based microspheres (first and second, third andfourth, columns in Table 1). The loading and loading efficiency in theNaCl-based microspheres with 1 h treatment were much lower than those inthe original material (third and fifth columns in Table 1), butincreased with time of NaCl treatment. The 15 h treatment led to theloading of about 0.15 μg of HRP per mg of silk microspheres, close tothe level in the original material (third and last columns in Table 1).It is likely that some empty lipid vesicles that were not yet fusedafter shorter NaCl treatments were co-precipitated with silkmicrospheres, which contributed to the measured weights and lowered theloading as a result.

3. HRP release from silk microspheres

HRP that was encapsulated in lyophilized DOPC-silk displayed asignificant release once the material was suspended in PBS buffer (FIG.7A). In contrast, less than 5% HRP was released from the MeOH-basedmicrospheres (with or without freeze-thaw treatment) into thesurrounding buffer over a period of one month (FIG. 7A). The activity inthe microspheres, however, dropped slowly, with about 50% remainingafter one month (data not shown). NaCl-based microspheres releasedencapsulated HRP at different release rates, depending on the NaCltreatment time used. When the treatment lasted for 15 h, a sustainedrelease which reached maximal level after 15 days was achieved (FIG.7B). The 1 h and 4 h treated samples released HRP more quickly. For allthese three samples, the HRP release reached about 200%. It is knownthat HRP activity can be inhibited by many factors, including metal ionslike Mn²⁺, Co²⁺, Ni²⁺, and Cu²⁺, L-cystine and sulfide, and surfactantsand lipids. Therefore, it is likely that some of these factors withinthe microspheres inhibited certain HRP activity, resulting in anunderestimation of HRP loading (Table 1). Once released to the buffer,the inhibited HRP activity was restored.

The HRP release as calculated by dividing the amount of release by theloading, which produced values higher than 100%. It is hard to determinethe absolute HRP loading in this case since it is difficult to extractHRP from silk microspheres while keeping the enzyme active. NaCltreatment induces the formation of β-sheet structures, as demonstratedin FIG. 5, with beta sheet content dependent on time of treatment, whichproduced different drug release profiles.

The encapsulated HRP was released slower from MeOH-based microspheres ascompared to NaCl-based microspheres with 15 h treatment, despite thefact that their β-sheet contents were both high (58% and 67%,respectively). The discrepancy might be due to the different amount ofphospholipids in MeOH-based microspheres (1%) and NaCl-basedmicrospheres (17%). It is believed that having more phospholipids inmicrospheres provided more channels for HRP to escape.

Because of its excellent entrapment capability, MeOH-based silkmicrospheres are the preferred long-term drug delivery and enzymeimmobilizations. It is believed that other alcohol- or solvent-basedsilk microspheres, such as ethanol, propanol, acetone, chloroform, orpolyethylene glycol solutions, would provide similar entrapmentcapabilities for drug delivery. Because of its mild preparationcondition and controllable crystalline β-sheet structure formation,NaCl-based microspheres are the preferred microspheres for thoseapplications in which protein drugs or other therapeutic drugs aresusceptible to methanol or alcohol treatment alternative drug releasekinetics are needed. It is believed that other salt-based silkmicrospheres, such as potassium chloride, would also be suitable forthese applications.

The silk microspheres may also be used for tissue engineeringapplications. For instance, by combining silk scaffolds with themicrospheres, the system can be used to deliver growth factors in atime- and/or spatial-controllable manner so that the artificial tissueslike bone and cartilage can be generated with more localized controlfrom these scaffolds. Depending on the processing, MeOH-based andNaCl-based silk microspheres released encapsulated HRP with differentkinetics, suggesting that the silk microspheres can be useful and cancarry sufficient growth factors for tissue engineering applications.

Aside from controlled drug delivery, silk microspheres can also be usedto immobilize enzymes for biosensor purposes. For instance, by combiningsilk microspheres and layer-by-layer coating techniques using silkfibroin, enzyme-based electrodes can be envisioned for use in a varietyof applications, such as in the food industry, environmental control,and biomedical applications.

1. (canceled)
 2. A composition comprising silk fibroin microspheresdistributed in a silk fibroin matrix, wherein the silk fibroinmicrospheres comprise a therapeutic agent.
 3. The composition of claim2, wherein the silk fibroin matrix is a silk fibroin scaffold.
 4. Thecomposition of claim 2, wherein the silk fibroin matrix comprises atleast one silk fibroin layer.
 5. The composition of claim 2, wherein thesilk fibroin matrix is a hydrogel.
 6. The composition of claim 2,wherein the therapeutic agent is selected from the group consisting ofproteins, peptides, nucleic acids, peptide nucleic acids (PNAs),aptamers, antibodies, growth factors, cytokines, enzymes, smallmolecules, and any combinations thereof.
 7. The composition of claim 2,wherein the therapeutic agent comprises an antibiotic.
 8. Thecomposition of claim 2, wherein the therapeutic agent comprisesadenosine.
 9. The composition of claim 2, wherein the silk fibroinmicrospheres comprise a multilamellar-structured silk fibroinmicrosphere.
 10. The composition of claim 2, wherein the silk fibroinmicrospheres comprise a unilamellar-structured silk fibroin microsphere.